Differing impact of phosphoglycerate mutase 1-deficiency on brown and white adipose tissue

Summary Brown adipose tissue (BAT) is a metabolically active organ that contributes to the thermogenic response to cold exposure. In addition, other thermogenic cells termed beige adipocytes are generated in white adipose tissue (WAT) by cold exposure. Although activation of brown/beige adipose tissue is associated with mobilization of both glucose and lipids, few studies have focused on the role of glycolytic enzymes in regulating adipose tissue function. We generated mouse models with specific deletion of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) from adipose tissue. Deletion of Pgam1 from both BAT and WAT promoted whitening of BAT with beiging of visceral WAT, whereas deletion of Pgam1 from BAT alone led to whitening of BAT without beiging of WAT. Our results demonstrate a potential role of glycolytic enzymes in beiging of visceral WAT and suggest that PGAM1 would be a novel therapeutic target in obesity and diabetes.


INTRODUCTION
The number of patients with obesity or diabetes has increased dramatically in the modern world, and these conditions have become major health problems for many societies (Van Gaal et al., 2006;Yanovski and Yanovski, 2002). It has been suggested that a potential therapeutic strategy for diabetes and its complications could involve activation of brown adipose tissue (BAT), which normally consumes excessive calories by generating heat in response to cold exposure (Hankir et al., 2016). Cold stimulates the sympathetic nervous system to promote lipolysis and thermogenesis, and is believed to be the most potent activator of BAT. Several studies have demonstrated that chronic acclimation of adult humans to cold increases the amount of activated BAT in the supraclavicular region, and this change is associated with improvement of insulin sensitivity (Lee et al., 2014) or with an increase of cold-induced energy expenditure (van der Lans et al., 2013;Yoneshiro et al., 2013). However, activation of BAT is impaired by aging, obesity, and the diabetic state in humans and in animal models (Cypess et al., 2009;Rogers et al., 2012;Yoneshiro et al., 2011), although the underlying molecular mechanisms have not been fully elucidated.
Cold exposure also activates inducible thermogenic adipocytes in white adipose tissue (WAT). These cells, known as beige adipocytes, appear to possess specific regulatory machinery for a thermogenic response and have a distinct developmental origin from brown adipocytes (Harms and Seale, 2013;Wu et al., 2012). In mice, inguinal WAT (iWAT) is known to readily undergo ''beiging'' in response to cold exposure, whereas gonadal WAT (gWAT) shows a much weaker beiging response, presumably because of differential expression of transcription factors involved in brown/beige adipocyte differentiation (Kajimura et al., 2015). In humans, supraclavicular BAT has a similar molecular signature to that of mouse beige adipocytes (Lee et al., 2014;Sharp et al., 2012;Shinoda et al., 2015;Wu et al., 2012), whereas BAT at other sites expresses classical BAT markers (Cypess et al., 2013;Nagano et al., 2015;Xue et al., 2015). A number of research groups have identified factors that regulate the activity of BAT and/or beige adipose tissue and could be potential therapeutic targets (Reitman, 2017;Villarroya and Vidal-Puig, 2013). These molecules include a number of growth factors and cytokines, such as fibroblast growth factor 21 (Fisher et al., 2012), adiponectin (Hui et al., 2015), natriuretic peptides (Bordicchia et al., 2012), prostaglandins (Vegiopoulos et al., 2010), and irisin (Bostrom et al., 2012). Thyroid hormone and parathyroid hormone are also known as BAT activators (Kir et al., 2016;Lin et al., 2015). Moreover, various transcription factors (Ma et al., 2015;Tai et al., 1996) and signaling molecules (Bi et al., 2014;Whittle et al., 2012) were reported to be involved in activating BAT and beige adipose tissue. Although b 3 -adrenergic stimulation is critical for activating thermogenic  Activation of BAT is associated with increased mobilization of glucose and lipids, which are utilized for generation of heat, and therefore BAT acts as a metabolic sink for these energy sources (Bartelt et al., 2011;Seale et al., 2011). The interaction between lipid metabolism and activation of BAT has been extensively investigated. For example, studies involving genetic deletion of factors that promote fatty acid oxidation have revealed an essential role of these factors in BAT activation by cold exposure (Ji et al., 2008;Lee et al., 2015;Tolwani et al., 2005). Inhibition of intracellular triglyceride lipolysis suppresses cold-induced activation of BAT in humans and animals, but lipolytic activity in BAT itself is not essential because defective BAT lipolysis can be fully compensated by circulating fatty acids derived from WAT (Schreiber et al., 2017;Shin et al., 2017). It has also been reported that lipid biosynthesis is required for the thermogenic response of BAT because it modulates the expression of mitochondrial electron transport chain components (Tan et al., 2015). Accumulating evidence suggests a critical role of glycolysis in regulating adipose tissue function (Hankir and Klingenspor, 2018;Isidor et al., 2020;Jung et al., 2021;Nguyen et al., 2020), but it remains unclear how glycolytic enzymes differently act on various adipose tissues. Therefore, we investigated whether BAT and WAT were regulated by the glycolytic enzyme phosphoglycerate mutase (PGAM), which catalyzes conversion of 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG), by generating mice with adipose tissue-specific PGAM deficiency.

RESULTS
Adipose tissue-specific Pgam1 deletion has differing effects on BAT and WAT We first determined the level of Pgam expression in various mouse tissues. PCR revealed high levels of Pgam1 expression in the brain and BAT, as well as moderate Pgam1 expression in the WAT, aorta, liver, and kidney ( Figure 1A). Pgam2, a muscle-specific isoform, was predominantly expressed by cardiac and skeletal muscle ( Figure 1A). Then we generated adipose tissue-specific Pgam1 knockout mice (Adipoq-Cre; floxed Pgam1, Adipo-Pgam1 KO mice). PCR showed that Pgam1 expression was significantly reduced in the BAT of these mice, as well as in gonadal WAT (gWAT) and inguinal WAT (iWAT) ( Figures 1B and S1A). Western blot analysis also demonstrated significant downregulation of Pgam1 expression in BAT and WAT, along with reduced enzyme activity ( Figure 1C). Both body weight and food intake showed no differences between Adipo-Pgam1 KO mice and their littermate controls ( Figures 1D and 1E). Analysis of CT data revealed a decrease of total WAT weight in Adipo-Pgam1 KO mice ( Figure 1F). In these mice, BAT weight was increased and iWAT weight was reduced, whereas gWAT weight was unchanged ( Figures 1G, 1H, and S1B). Investigation of the metabolic profile of Adipo-Pgam1 KO mice demonstrated lower energy expenditure ( Figure 1I) and glucose intolerance/hyperinsulinemia compared with their littermate controls ( Figure 1J). Despite these metabolic abnormalities, Adipo-Pgam1 KO mice showed superior cold tolerance compared with littermate controls ( Figure 1K). Histological examination of Adipo-Pgam1 KO mice showed that the BAT of these animals contained larger lipid droplets and had undergone whitening, whereas gWAT and iWAT demonstrated an increase of beige adipocytes ( Figure 1L). We also found an increase of macrophage infiltration into WAT of Adipo-Pgam1 KO mice ( Figures S1C and S1D). These findings suggested that BAT dysfunction and iScience Article WAT inflammation induced by Pgam1 deficiency led to abnormal glucose metabolism, but beiging of WAT contributed to preservation of the thermogenic response.

Opposing metabolic profiles of BAT and WAT in Adipo-Pgam1 KO mice
To investigate the mechanisms underlying the phenotypic changes of BAT and WAT induced by Pgam1 deficiency, we conducted transcriptomic and metabolomic analyses of Adipo-Pgam1 KO mice and littermate controls. Transcriptomic analysis demonstrated marked downregulation of enzymes involved in glycolysis and the tricarboxylic acid (TCA) cycle in BAT from Adipo-Pgam1 KO mice compared with BAT from littermate controls (Figures 2A and 2B), corresponding to the observed whitening of BAT in the KO mice ( Figure 1L). In contrast, expression of these enzymes was upregulated in WAT from Adipo-Pgam1 KO mice, particularly gWAT, compared with WAT from littermate controls (Figures 2A, 2B, S2A, and S2B). Metabolomic analysis of the   S2A, and S2B). Gene ontology enrichment analysis indicated the development of an inflammatory response and metabolic dysfunction in BAT from Adipo-Pgam1 KO mice ( Figure S3). In contrast, WAT from these mice showed enhancement of metabolic activity, supporting the observed phenotypic changes of adipose tissue ( Figure S3).

Adipo-Pgam1 KO mice show whitening of BAT and beiging of WAT
To further investigate the adipose tissues changes in Adipo-Pgam1 KO mice, we performed histological examination by using uncoupled protein 1 (Ucp1) and carnitine palmitoyl transferase 1b (Cpt1b) as markers for thermogenic adipocytes. Compared with their littermate controls, the number of Ucp1-positive cells was significantly decreased in BAT from Adipo-Pgam1 KO mice, whereas it was significantly increased in gWAT from these mice ( Figure 3A). Likewise, there were fewer Cpt1b-positive cells in BAT from Adipo-Pgam1 KO mice and more Cpt1b-positive cells in gWAT from these mice than in the corresponding tissues from littermate controls ( Figure 3A). PCR revealed upregulated expression of Ucp1 and Cpt1b in gWAT from Adipo-Pgam1 KO mice, whereas expression was downregulated in BAT ( Figure 3B). Similar results were observed in expression of other beige makers ( Figure S4). It is interesting that iWAT showed milder beiging than gWAT in these mice after Pgam1 deletion, including a smaller increase of Ucp1 or Cpt1b-positive cells and less marked upregulation of beige adipocyte markers ( Figures S5A-S5C). In Adipo-Pgam1 KO mice, electron microscopy detected a number of mitochondria with structural collapse in BAT, whereas there was an increase of normal mitochondria in gWAT ( Figure 3C) and also in iWAT to a lesser extent ( Figure S5D). We also observed a significant increase of TUNEL-positive cells in BAT from Adipo-Pgam1 KO mice ( Figure 3D). Norepinephrine was not elevated in plasma or in WAT from Adipo-Pgam1 KO mice ( Figure 3E), suggesting that beiging of gWAT after Pgam1 deletion was not mediated by neural activation because of BAT dysfunction.

BAT-specific Pgam1 deletion does not promote beiging of WAT
It is possible that dysfunctional BAT promotes beiging of WAT via the central nervous system. To further investigate the mechanisms underlying Pgam1 deletion-induced beiging of gWAT, we generated BAT-specific Pgam1 knockout mice (Ucp1-Cre; floxed Pgam1, BAT-Pgam1 KO mice). In these mice, Pgam1 expression was significantly reduced in BAT, but not in WAT ( Figure 4A). Body weight and food intake did not differ between BAT-Pgam1 KO mice and their littermate controls ( Figures 4B and 4C). BAT-Pgam1 KO mice showed a significant increase of BAT weight, whereas there were no changes of gWAT or iWAT weights ( Figure 4D). Targeted deletion of Pgam1 from BAT led to impairment of cold tolerance with normal glucose and insulin tolerance ( Figures 4E and 4F). We found that BAT-Pgam1 KO mice demonstrated lower energy expenditure compared with their littermate controls ( Figure 4G), although the difference was not statistically significant. Histological examination revealed that deletion of Pgam1 from BAT led to whitening of BAT, but did not affect WAT ( Figure 5A). In comparison with their littermate controls, electron microscopy showed that Pgam1 deletion was associated with an increase of abnormal mitochondria in BAT from Adipo-Pgam1 KO mice, but no specific changes were observed in WAT ( Figure 5B). Although Ucp1-positive cells and Cpt1b-positive cells were decreased in BAT from BAT-Pgam1 KO mice, these cells were not increased in WAT ( Figure 5C). According to PCR analysis, there was downregulation of thermogenic adipocyte markers in BAT from BAT-Pgam1 KO mice compared with littermate controls, whereas there was no difference of these markers in WAT ( Figure 5D). Taken together, these findings suggested that Pgam1 deletion directly induces beiging of gWAT.
To test this hypothesis, we crossed Adipo-Pgam1 KO mice with EGFP Cre reporter mice (Adipoq-Cre; floxed Pgam1; CAG-LSL-EGFP). In these mice, expression of EGFP and Cpt1b was co-localized in gWAT ( Figure S6A). We also found that Cre protein immunoreactivity was co-localized with that of Cpt1b in gWAT ( Figure S6B), iScience Article suggesting specific upregulation of Cpt1b expression in gWAT cells with Pgam1 deletion. To further examine whether Pgam1 deletion induces beiging of gWAT, we conducted single cell RNA sequence analysis of the stromal vascular fraction from gWAT of Adipo-Pgam1 KO mice and their littermate control mice. We identified a cluster in the fibroblast cluster of gWAT of littermate control mice that was rich for adipocyte markers (Fibro-blast4; Figure S6C), suggesting that this population were preadipocytes and that Pgam1 was deleted in this population of Adipo-Pgam1 KO mice by the Cre recombination driven by the Adiponectin promoter. Consistent with this idea, the corresponding cell population in Adipo-Pgam1 KO mice showed a marked downregulation of Pgam1 expression. Analysis of the differential expressed genes in this cluster between genotypes revealed upregulation of genes indicative of beige cells ( Figure S6D), suggesting a causative relationship between Pgam1 deletion and beiging of gWAT.

Inhibition of glycolysis increases beige-like cells in adipocytes
Gene set enrichment analysis indicated that there was marked downregulation of the degradation of branched amino acids (BCAA) in gWAT from Adipo-Pgam1 KO mice ( Figure S3). In agreement with this finding, metabolomic analysis showed accumulation of various amino acids including BCAA in gWAT from these mice ( Figure 6A). It is well accepted that an increase of branched amino acids leads to activation of the mTor pathway and inhibition of autophagy (Nie et al., 2018). We previously reported that dietary obesity promotes the whitening of BAT by activating mitophagy (Shimizu et al., 2014). In addition, Altshuler-Keylin et al. demonstrated that autophagy-mediated clearance of mitochondria is required for beige-to-white adipocyte phenotypic reversal and that inhibition of autophagy leads to maintenance of functioning beige adipocytes (Altshuler-Keylin et al., 2016). In agreement with our metabolomic data, in the present study we found upregulation of the mTor pathway in gWAT from Adipo-Pgam1 KO mice (Figure 6B); this upregulation was associated with increased expression of p62 ( Figure 6C). We also examined BAT tissues and plasma from Adipo-Pgam1 KO and/or BAT-Pgam1 KO mice and found no significant changes in BCAA-mTor-autophagy signals ( Figures S7A-S7E).
To examine a relationship between Pgam1 deletion and mTor-induced beiging in differentiated adipocytes, we infected NIH3T3L1 cells with an AAV vector that codes short hairpin (sh)-Pgam1. Consistent with the in vivo data from Adipo-Pgam1 KO mice, deletion of Pgam1 from differentiated adipocytes led iScience Article to an increase in BCAA levels and activation of mTor signaling ( Figures S8A-S8C). Pgam1 deletion upregulated expression of Ucp1 ( Figure S8D), and this upregulation was attenuated by rapamycin ( Figure S8E). Treatment of differentiated adipocytes with BCAA led to upregulation of Ucp1 ( Figure S8F). Likewise, treatment of differentiated adipocytes with MHY1485, a potent mTOR activator that also inhibits autophagy, increased expression of p62 and Ucp1 (Figures S8G and S8H).

DISCUSSION
The present study demonstrated that deletion of Pgam1 from BAT and WAT had a different impact on each tissue, because it promoted whitening of BAT and beiging of WAT without any change of adipose tissue catecholamine levels. Deletion of Pgam1 from BAT alone did not affect WAT, excluding the possibility that BAT dysfunction induced beiging of WAT via activation of the sympathetic nervous system. These findings were further supported by in vitro experiments using differentiated adipocytes, which showed that deletion of Pgam1 led to an increase of beige cell makers, as well as by histological analysis of EGFP reporter mice. Taken together, our results indicate that beiging of WAT is induced by deletion of Pgam1 via a cell-autonomous mechanism.
It has been reported that Pgam1 has a role in tumorigenesis and cellular aging, and Pgam1 has been suggested as a therapeutic target for age-associated diseases including cancer (Hitosugi et al., 2012;Jiang et al., 2014;Kondoh et al., 2005). Interestingly, we observed upregulation of beige makers and downregulation of glycolytic enzymes in gWAT of aged mice with BAT whitening (data not shown), suggesting that inactivation of glycolysis may be involved in regulating the adaptive response of gWAT to age-associated BAT dysfunction. This adaptive response may provide a survival advantage for aged animals by maintaining minimal thermogenesis in response to cold and preventing inappropriate energy consumption under starvation conditions despite age-associated whitening of BAT. It is known that cold exposure preferentially promotes the beiging of iWAT rather than gWAT, whereas we found that Pgam1 deletion particularly increased beige-like cells in gWAT. Because visceral fat has a pathological role in metabolic dysfunction leading to type 2 diabetes, inhibition of glycolysis in gWAT could be an attractive strategy for treating metabolic diseases like type 2 diabetes.
In gWAT from Adipo-Pgam1 KO mice, we found accumulation of branched chain amino acids along with upregulation of the synthetic enzymes. Branched chain amino acids are the most abundant essential amino acids, and are not only substrates for the synthesis of nitrogenous compounds, but also act as signaling molecules in the regulation of glucose, lipid, and protein metabolism or immunity via special signaling networks such as the mTor pathway (Nie et al., 2018). Consistent with these reports, we identified activation of the mTor pathway in gWAT from Adipo-Pgam1 KO mice, which was associated with inhibition of autophagy and an increase of beige-like cells. Our transcriptomic analysis also demonstrated upregulation of the phosphoinositide 3-kinase/Akt pathway in gWAT from Adipo-Pgam1 KO mice that could possibly account for activation of mTor signaling (the Gene Expression Omnibus database GSE186030). When Halama et al.
(2016) investigated the crucial metabolic pathways for differentiation of white adipocytes, their transcriptomic and metabolomic analyses identified phosphatidylcholine synthesis, fatty acids metabolism, and catabolism of branched chain amino acids as key regulatory pathways. They also demonstrated that a metabolic switch from catabolism of branched chain amino acids to lipid synthesis was crucial for white adipocyte differentiation. Taken together with our results, these findings suggest that branched chain amino acids may have a role in the regulation of white and beige adipocyte differentiation.
Our in vitro experiments using differentiated adipocytes suggested that autophagy could be involved in regulating the beiging of white adipocytes, but it remains to be determined whether Pgam1 deletion actually inhibits autophagy to promote the beiging response of gWAT. It would be interesting to examine the effect of specific mTor pathway activation (for example, using floxed Tsc1/2 mice) in gWAT of Adipo-Pgam1 KO mice, although a visceral WAT-specific Cre driver mouse is not available at present. Indeed, we still do not fully understand how Pgam1 deletion activates the mTor pathway, how Pgam1 deletion promotes accumulation of branched chain amino acids, or whether accumulation of these amino acids underlies activation of the mTor pathway and beiging of gWAT in Adipo-Pgam1 KO mice. Thus, further studies will be required to develop novel therapeutic strategies for metabolic disease based on modulation of cell metabolism in adipose tissue.

Limitations of the study
The results of the present study suggest that the BCAA-mTor-autophagy axis may be involved in beiging of gWAT of Adipose-Pgam1 KO mice, but a causative relationship remains to be determined.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:     , Li, Y., Takahashi, H., et al. (2015). Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes. Nat.

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Tohru Minamino at t.minamino@juntendo.ac.jp.

Materials availability
This study did not generate new unique reagents.

Data and code availability
All data reported in this paper will be shared by the Lead contact upon request. Gene expression data are available through the Gene Expression Omnibus database (GSE186030, GSE200374).

Animal models
All of the animal experiments were conducted in compliance with the protocol reviewed by the Institutional Animal Care and Use Committee of Niigata University and approved by the President of Niigata University. Adipoq-Cre mice (Eguchi et al., 2011) were purchased from the Jackson Laboratory (ME, U.S.A.). Adipoq-Cre or Ucp1-Cre (C57BL/6J background) (Kong et al., 2014) were crossed with mice carrying floxed Pgam1 alleles with a C57BL/6 background to generate mice with adipose tissue-(Adipo-Pgam1 KO) or brown adipose tissue (BAT)-(Ucp1-Pgam1 KO) specific knockout of Pgam1. The genotypes of the littermate controls were Adipoq-Cre -: Pgam1 flox/flox or Ucp1-Cre -: Pgam1 flox/flox . CAG-LSL-EGFP mice were provided by RIKEN (RBRC09806) (Kawamoto et al., 2000). All experiments were performed using male mice.

Cell culture
The mouse pre-adipocyte cell line, NIH-3T3L1 cells were cultured in high glucose DMEM (Gibco, 12430) with 10% fetal bovine serum (FBS) and 100U/mL penicillin/streptomycin solution (P/S), and differentiation was induced by adding 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone, and 5 mg/mL insulin (Sigma Aldrich) for 48 h. Fully differentiated adipocytes were used for further analysis after 7 days of culture. iScience Article